Membrane proteins are responsible for many important properties and functions of biological systems: they transport ions and molecules across the membrane, they act as receptors, and they have roles in the assembly, fusion, and structure of cells and viruses. Despite the abundance and clear importance of membrane-associated molecules, very little structural information about these systems exists. The objective of the proposed research is to develop new EPR structural biology methods to probe the structural and dynamic properties of integral membrane proteins. The novel experiments proposed in this study will reveal new insights concerning the structural characteristics of phospholamban (PLB) in lipid bilayers and develop new biophysical methods utilizing bicelles. The NIH has recognized the importance of studying the structural properties of integral membrane proteins with PA-06-119. This program announcement has specifically requested for new biophysical techniques to probe the structures of membrane proteins. In accordance with this program announcement, new EPR spectroscopic methods will be developed to probe the structures of integral membranes and directly compare our results with solid-state NMR spectroscopic data. This proposal is method development in nature;thus, the integral membrane protein PLB will be used a model membrane protein system. We feel that this new approach will move the field forward so that researchers can more easily and inexpensively determine the structural topology of integral membrane proteins using spin-label EPR spectroscopy. The specific aims of this proposal consist of the following: (1) Develop a new spin- label EPR spectroscopy technique to determine the helical tilt of integral membrane peptides inside bicelles using PLB as a model;(2) Develop alternative membrane protein alignment methods using mechanically oriented phospholipid bilayers and phospholipid bilayer nanotube arrays using spin-label EPR spectroscopy;(3) Determine the orientation and secondary structure of phospholamban with respect to the phospholipid bilayer using solid-state NMR spectroscopy;and (4) Investigate the membrane-bound protein dynamics of phospholamban with spin-label EPR spectroscopy and solid-state NMR spectroscopy.Project Narrative The NIH has recognized the importance of studying the structural properties of integral membrane proteins with PA-06-119. This program announcement has specifically requested for new biophysical techniques to probe the structures of membrane proteins. In accordance with this program announcement, we will develop new EPR spectroscopic methods to probe the structures of integral membranes and directly compare our results with solid-state NMR spectroscopy to validate this new approach. This proposal is method development in nature;thus, PLB will be used as a model membrane protein system.